Structural changes in oocyte nucleoli ofXenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II–III oocytes and within the nucleoli of relatively quiescent stage VI oocytes of Xenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II–III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II–III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4′,6-diamidino-2-phenylindole showed condensed DNA within nucleolar FCs of both stage II–III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble. Received: 18 March 1996 / Accepted: 16 April 1996     

Subcellular Distribution of Distinct Nucleolin Subfractions Recognized by Two Monoclonal Antibodies

Monoclonal antibodies binding to different domains of nucleolin have been used to localize nucleolin in tissue culture cells ofXenopus laevis.The monoclonal antibody b6-6E7 binds to an epitope in the N-terminal domain, which contains arrays of phosphorylation consensus sites. This monoclonal antibody binds to nucleolin of oocytes and of eggs with high affinity. In contrast, the monoclonal antibody Nu-1H6 binds poorly to the modified forms of nucleolin arising during meiosis and mitosis. In interphase cells, monoclonal antibody b6-6E7 preferentially stains the periphery of the nucleoli, where most of the rRNA accumulates. Staining by monoclonal antibody Nu-1H6 complements this pattern by staining mainly the center of the nucleoli. The epitope of monoclonal antibody Nu-1H6 is within the central domain of nucleolin, which contains the first two RNA binding domains. RNase treatment of cells results in loss of nucleolin from nucleoli. In mitotic cells, both monoclonal antibodies decorate the surface of condensing chromosomes in prophase. The periphery of the condensed chromosomes in metaphase and anaphase is preferentially stained by monoclonal antibody b6-6E7.     

In vitro binding of nucleolin to double-stranded telomeric DNA

We have purified a 100 kDa protein, resolved in a Southwestern binding screen of total nuclear proteins from Hela cells with double-stranded human telomeric probe. A polyclonal antiserum raised by this protein recognizes purified nucleolin and stains nucleoli in growing Hela cells. We demonstrate that a truncated form of human nucleolin and a purified deletion derivative of mouse nucleolin bind in vitro to duplex telomeric DNA. This study suggests a new link between telomeres and the nucleolus.     

Internalization of anti-nucleolin antibody into viable HEp-2 cells

Anti-nucleolin antibodies have been detected in patients with systemic connective tissue diseases (SCTD) including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). In vivo bound autoantibodies to nucleoli of epidermal keratinocytes have been demonstrated in skin from patients with SCTD. In this study, monoclonal antibody to nucleolin (D-3) was used to determine the distribution of nucleolin in different culture cells including HEp-2, HepG2, HRCC, Molt-4 and Wil2 cells. Nucleolin was found to be present on the surface of HEp-2 and HepG2 cells, but not on the surface of HRCC and lymphoblastoid (Molt-4 and Wil2) cells; in contrast, nucleolin was detected in the nucleoli of all permeabilized cells examined. In immunoprecipitation, using extracts from ³²P-labeled HEp-2 cells as antigenic source, cell membrane as well as nuclear nucleolins were found to be phosphorylated with a molecular weight of 105 kDa. Viable HEp-2 and HepG2 cells were cocultured with IgG fraction of D-3 in a CO₂ incubator for 1 to 24 h, and then permeabilized with acetone followed by immunofluorescence staining with FITC-labeled goat anti-mouse IgG antibodies. Nucleolar staining was observed in cells after 10 h or longer of coculture. These data indicated that D-3 antibody reacted with cell membrane nucleolin and subsequently gain access into cells in a process related to pinocytosis.     

Mucosal antibody responses of colonized cattle to Escherichia coli O157-secreted proteins, flagellin, outer membrane proteins and lipopolysaccharide

The aim of this work was to characterize adaptive mucosal immune responses to Escherichia coli O157:H7 at the principal site of colonization in the bovine species. Following experimental infection, extracts from terminal rectum mucosal samples were tested for IgA antibodies by immunoblotting against different bacterial antigens including: whole-cell E. coli O157:H7 with and without proteinase treatment, outer membrane and cytoplasmic preparations, secreted protein supernatants and purified E. coli O157 lipopolysaccharide and H7 flagellin. Lipopolysaccharide and H7 flagellin preparations were also used to coat enzyme-linked immunosorbent assay plates to determine mucosal IgG1 and IgA antibody titers. In this work, evidence is presented of strong local IgA immune responses induced following infection at the bovine terminal rectal mucosa directed against multiple antigens including type III secretion-dependent proteins, O157 lipopolysaccharide, H7 flagellin and OmpC.     

Nucleolar proteins identified in human cells as antigens by sera from dogs with autoimmune disorders

In the course of a systematic screening of sera from dogs suffering from autoimmune disorders, three sera were shown by indirect immunofluorescence to characteristically label the nucleoli and nucleoplasm of human cell lines (Hep-2 and HeLa). This pattern of staining persisted throughout the cell cycle, except for mitosis when the fluorescence was localized in extrachromosomal areas. By immunoblotting nuclear and subnuclear fractions, three polypeptides of 110,000, 95,000, and 45,000 Da apparent molecular weight were identified, which reacted with all three sera. By means of affinity purification, it was shown that an antibody specific for any one of the three proteins also reacts with the two others. This antigenic cross-reactivity suggested regions of structural homology shared by the three proteins. Indeed, treatment of nucleoli with high concentrations of DNase I containing residual proteolytic activity resulted in the disappearance of the 110- and 95-kDa proteins and the concomitant appearance of a doublet of 45-kDa proteins. Subnuclear localization studies indicated that all three polypeptides were located in both nucleoli and nucleoplasm. Significantly, the 110-kDa protein differs from the major nucleolar protein, nucleolin, by its electrophoretic mobility in two-dimensional gels, its location in nucleoli and in nucleoplasm, its absence in nucleolar organizer regions of chromosomes, and its differential solubility of DNase I. Therefore, the three antigenically related species reported in this study constitute a new class of nucleolar proteins.     

The chemical composition of nuclei and chromosomes isolated by the Langmuir trough technique

The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed.     

The Murine Nucleolin Protein Is an Inducible DNA and ATP Binding Protein Which Is Readily Detected in Nuclear Extracts of Lipopolysaccharide-Treated Splenocytes

A 100-kDa DNA binding protein was found to be dramatically up-regulated upon the mitogenic stimulation of murine splenocytes with bacterial lipopolysaccharide (LPS). The induced DNA binding protein was also found to exhibit moderate binding specificity for the immunoglobulin isotype switch DNA repeats. Furthermore, the induction of the 100-kDa protein by LPS was found to be mediated by both an increase in the protein's stability and an increase in the synthesis of the protein. In vitro phosphorylation experiments revealed that the 100-kDa DNA binding protein was one of the most heavily phosphorylated proteins in both lymphoid and nonlymphoid nuclear extracts. Although this in vitro phosphorylation initially appeared to be mediated by a potent nuclear kinase activity, it was later determined that a significant part of the detected labeling was due to the direct binding of ATP by the 100-kDa protein. Antibodies raised to the 100-kDa DNA binding protein were used to isolate cDNA clones from a lymphocyte cDNA λgt11 expression library. Nucleotide sequence analysis revealed that the cloned cDNAs were identical to the mouse nucleolin gene. The β-galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and a more severely truncated 45-kDa protein (encoded by exons 5-14 of nucleolin) were both found to bind strongly to DNA and ATP. Furthermore, the strength of DNA binding was found to be highly dependent on the overall dG content of the DNA probes. Our experiments also revealed that apart from binding ATP and G-rich DNA, nucleolin directly bound GTP, dATP, and dGTP, but not dCTP, dTTP, or dUTP. Computer analysis revealed that the putative ATP binding domains appear to fall within two of the phylogenetically conserved RNA binding domains of nucleolin.     

The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.     

CHROMOSOMAL LOCALIZATION OF REPETITIVE DNA IN THE NEWT, TRITURUS

The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization     

Cleavage of nucleolin and argyrophilic nucleolar organizer region associated proteins in apoptosis-induced cells

To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.     

Alzheimer's amyloid beta-peptide (1-42) induces cell death in human neuroblastoma via bax/bcl-2 ratio increase: an intriguing role for methionine 35

Nucleolin associates with various DNA repair, recombination, and replication proteins, and possesses DNA helicase, strand annealing, and strand pairing activities. Examination of nuclear protein extracts from human somatic cells revealed that nucleolin and Rad51 co-immunoprecipitate. Furthermore, purified recombinant Rad51 associates with in vitro transcribed and translated nucleolin. Electroporation-mediated introduction of anti-nucleolin antibody resulted in a 10- to 20-fold reduction in intra-plasmid homologous recombination activity in human fibrosarcoma cells. Additionally, introduction of anti-nucleolin antibody sensitized cells to death induced by the topoisomerase II inhibitor, amsacrine. Introduction of anti-Rad51 antibody also reduced intra-plasmid homologous recombination activity and induced hypersensitivity to amsacrine-induced cell death. Co-introduction of anti-nucleolin and anti-Rad51 antibodies did not produce additive effects on homologous recombination or on cellular sensitivity to amsacrine. The association of the two proteins raises the intriguing possibility that nucleolin binding to Rad51 may function to regulate homologous recombinational repair of chromosomal DNA.     

NopA64, a novel nucleolar phosphoprotein from proliferating onion cells, sharing immunological determinants with mammalian nucleolin

Five major soluble nuclear proteins associated with cell proliferation were identified in Allium cepa L. root cells. One of them, of 64 kDa, was revealed by Western blotting with anti-mammalian nucleolin antibodies. A polyclonal antibody raised against this protein, which we have named NopA64, localised it in the nucleolus as well as in nuclear coiled bodies. Together with NopA64, the antibody also revealed a smaller form, called NopA61. Both proteins were present in the soluble ribonucleoprotein fraction and in the nuclear matrix of proliferating cells, but NopA61 was the only form revealed in differentiated cells. NopA64 contained epitopes also present in other plants, in mammalian nucleolin and in its yeast homologue, gar2. In mammals, the highest homology was with 50-kDa nucleolin fragments containing the RNA-binding motifs and the glycine-arginine-rich (GAR) domain. NopA64 was moderately phosphorylated in vitro by exogenous casein kinase II and cdc2 kinase, whereas NopA61 was highly phosphorylated by casein kinase II. Furthermore, NopA61 was the only band detected after dephosphorylation as well as after endoproteolysis of NopA64. This protein could be one of the various functional homologues of mammalian nucleolin in plant cells. Received: 2 July 1996 / Accepted: 15 October 1996